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Article history: Hydrogenase (Hyd) activity and H2 production by Escherichia coli were studied at a low pH.
Received 19 September 2010 H2 production at pH 5.5 under glycerol fermentation was shown to be w1.5-fold higher
Received in revised form than that at pH 6.5 or above but less than that under glucose fermentation. It was inhibited
25 December 2010 by N,N0 -dicyclohexylcarbodiimide: H2 production inhibition was increased with decreasing
Accepted 29 December 2010 pH and almost maximal inhibition was observed at pH 5.5. The data on H2 production by
Available online xxx single and double mutants with defects in different Hyd-enzymes and in fhlA gene suggest
that under glycerol fermentation at a low pH, Hyd-1, Hyd-2 and Hyd-4 were operating in
Keywords: a reversed, non-H2 producing mode. Moreover, a role of fhlA gene in Hyd-3 and Hyd-4
Hydrogenases activity in H2 production is proposed under glucose fermentation at a low pH.
Glycerol fermentation ª 2011 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.
pH
Hydrogen production
Escherichia coli
Please cite this article in press as: Trchounian K, et al., Escherichia coli hydrogenase activity and H2 production under glycerol
fermentation at a low pH, International Journal of Hydrogen Energy (2011), doi:10.1016/j.ijhydene.2010.12.128
2 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 1 ) 1 e9
a Resistant to Kan.
Please cite this article in press as: Trchounian K, et al., Escherichia coli hydrogenase activity and H2 production under glycerol
fermentation at a low pH, International Journal of Hydrogen Energy (2011), doi:10.1016/j.ijhydene.2010.12.128
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 1 ) 1 e9 3
Whole cells for assay were prepared as described [9,18]. For shown). This seems to be in good conformity with data about
N,N0 -dicyclohexylcarbodiimide (DCCD) (Sigma, USA) inhibi- requirement of rich nutrients in the medium for glycerol
tion studies, cells were incubated with DCCD at 0.5 mM for fermentation by E. coli at acidic pH, whereas low supplemen-
10 min at 37 C. Dry weight of bacteria was determined as tation (as peptone) allowed glycerol fermentation at alkaline
previously [9]. pH [2,4]. However, mechanisms of this phenomenon have
been not clear yet [4].
2.3. Redox potential determination and hydrogen Eh, measured by Pt-electrode in the suspension of E. coli
production assays BW25113 wild type grown in peptone medium on glycerol
added at a low pH, washed and transferred into the assay
Redox potential (Eh) in bacterial suspension was measured medium, was decreased in the absence of glycerol, but this
using the oxidation-reduction, a titaniumesilicate (TieSi) (EO- change was negligible (Fig. 1, dotted curves). However upon
02, Gomel State Enterprise of Electrometric Equipment addition of glycerol (10 ml/l), Eh dropped markedly from
(GSEEE), Gomel, Belarus) and platinum (Pt) (EPB-1, GSEEE, or positive values (þ146 8 mV at pH 6.5 or þ326 18 mV at pH
PT42BNC, Hanna Instruments, Portugal) electrodes [9,18,32].
In contrast to TieSi-electrode measuring the overall Eh, a Pt-
electrode is sensitive to H2 under anaerobic conditions (in the
absence of O2) [33] allowing detection of H2 production.
Therefore, H2 production rate (VH2 ) was calculated through the
difference between the initial rates of decrease in Pt- and
TieSi-electrode readings per time and expressed as mV of Eh
per min per mg dry weight of bacteria as described before
[9,18,23,34,35]. Eh measurement by means of Pt- and TieSi-
electrodes was done in the assay buffer where bacterial
suspension was transferred and upon glycerol or glucose
addition mentioned. No significant differences between Pt-
and TieSi-electrodes readings were detected in bacterial
suspension without glycerol and glucose added; bacterial
count alteration in the suspension by w8e10-fold had no
marked effect on Eh value. This determination is closed to the
method with Clark-type electrode employed by Noguchi et al.
[26]: a correlation between Eh and H2 production was shown.
Note, the electrochemical measurement was reviewed to be
indirect for H2 production determination but can give accurate
and reproducible data [5,6].
H2 production was verified by the chemical assay based on
the bleaching of KMnO4 solution in H2SO4 with H2 [34]. This
method was suggested for detecting enhanced H2 production
[36]. Using the Durham tube method [18], H2 production
during E. coli growth was also estimated by the appearance of
gas bubbles in the test tubes over the bacterial suspension.
Please cite this article in press as: Trchounian K, et al., Escherichia coli hydrogenase activity and H2 production under glycerol
fermentation at a low pH, International Journal of Hydrogen Energy (2011), doi:10.1016/j.ijhydene.2010.12.128
4 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 1 ) 1 e9
Please cite this article in press as: Trchounian K, et al., Escherichia coli hydrogenase activity and H2 production under glycerol
fermentation at a low pH, International Journal of Hydrogen Energy (2011), doi:10.1016/j.ijhydene.2010.12.128
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 1 ) 1 e9 5
hyaB and hybC mutants and similar to that of the wild type: the
140 hybC latter was unexpected (Fig. 3). At pH 5.5 VH2 by wild type was
120 hyaB hybC higher (see Figs. 3 and 1), however it was lowered w1.3 and
100 w1.4-fold with the hyaB and hybC mutants, respectively; the
80 low rate was defined with the double hyaB hybC mutant (Fig. 3).
The results obtained suggest that, during glycerol fermenta-
60
tion at a low pH, increased H2 production was probable in the
40
absence of H2 uptake activity so as Hyd-1 and Hyd-2 were
20 working in a reversed H2 oxidizing mode and the other Hyd-
0 enzymes were responsible for H2 production (Fig. 4). Thus,
pH 7.5 pH 6.5 pH 5.5 recycling of produced H2 is required for hydrogen metabolism
during glycerol fermentation by E. coli. The latter and changed
VH2 by double hyaB hybC mutant are likely to the results
B 260 obtained by Redwood et al. [16] about compensatory uptake
240
function during H2 production under different conditions.
220 wild type
H2 production rate , %
To establish a role of Hyd-enzymes required for H2 production Fig. 4 e Different H2 producing and H2 uptaking Hyd-
during glycerol fermentation at a low pH, mutants with enzymes expressed by E. coli under glycerol fermentation
defective Hyd-1 and Hyd-2 (see Table 1) were studied. at pH 7.5 (A) or pH 5.5 (B). VH2 is H2 producing rate by whole
A decrease in Eh down to strong negative values was cells; V(Hyd) is H2 producing or H2 oxidizing rate by
observed with JW0955, JW2962 and MW1000 mutants (see appropriate Hyd-enzyme. Arrows are for direction of
Table 1) at pH 6.5 and 5.5 both (see Fig. 1). This indicates a high enzyme operation to produce and/or to oxidize H2. The
total Hyd-activity and H2 production under the conditions mode for Hyd-enzymes functioning at pH 5.5 during
mentioned above. In contrast to pH 7.5 when VH2 by single glucose fermentation is similar with that under glycerol
hyaB and hybC mutants was less than that with wild type, at fermentation (B). For the others, see the text.
Please cite this article in press as: Trchounian K, et al., Escherichia coli hydrogenase activity and H2 production under glycerol
fermentation at a low pH, International Journal of Hydrogen Energy (2011), doi:10.1016/j.ijhydene.2010.12.128
6 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 1 ) 1 e9
produced H2 could be proposed. This seems to be likely to the 3.4. Construction of E. coli strains deleted for fhlA gene
conclusion of Murarka et al. [4] about recycling of H2 evolved and defective in Hyd-3 and Hyd-4
by the FHL complex during glycerol fermentation and, in
addition, to the suggestion by Zbell and Maier [45] about a role To understand the mechanism of H2 production from glycerol,
of different levels of Hyd-1 to recycle all H2 production in we constructed the double mutant E. coli SW1002 ( fhlA hycG)
Salmonella enterica under fermentative conditions. The find- defective in FhlA and in Hyd-3 large subunit and SW1001 ( fhlA
ings above are also in accordance with the idea of Lukey et al. hyfG) defective in FhlA and Hyd-4 large subunit (see Table 1).
[17] about two-directional activity of Hyd-2 but under different The strains were verified via PCR; the PCR products using
conditions. primers flanking hycG have the expected size for BW25113
Moreover, at pH 6.5 double hyaB hybC deletions in E. coli (Fig. 5, A, lane 1), for the hycG mutant JWD2689 after deletion of
may affect the other Hyd-enzymes expression and activity the Kan resistance marker from JW2689 (Fig. 5, A, lane 2), and
since a pleiotropic effect of hyb genes deletions is suggested at SW1002 (Fig. 5, A, lane 3). As expected, no PCR product was
a low pH [46]. Alternatively but with less chance, double hyaB obtained for BW25113 (Fig. 5, A, lane 5) using a forward primer
hybC deletions resulting in the lack of large subunits of Hyd-1 upstream of fhlA and the backward primer inside the Kan
and Hyd-2 both in the appropriate Hyd complexes in the E. coli resistance marker, but the expected product size was obtained
membrane (see Table 1) could affect protein organization for SW1002 (Fig. 5A, lane 6).
within the membrane and induce conformational changes in PCR products of the expected size were also obtained using
appropriate Hyd-enzymes changing the total H2 producing primers flanking hyfG for BW25113 (Fig. 5, B, lane 1), the hyfG
activity. Interestingly, this is in favor with the suggestion that mutant including the Kan resistance marker JW2472 (Fig. 5B,
low level or not proper localization of Hyd-2 or other Hyd- lane 2), the hyfG mutant after deletion of the Kan resistance
enzyme can result in different coupling of H2 oxidation to FHL marker JWD2472 (Fig. 5, B, lane 3) and four independent
too [45]. In all cases, it is difficult to give a clear explanation for colonies of SW1001 (Fig. 5, B, lanes 4e7). The insertion of the
the finding on lower VH2 in double hyaB hybC mutant since Kan resistance marker at the fhlA locus of SW1001 was also
expression and regulation of Hyd-enzymes are different and verified for 4 independent colonies (Fig. 5, B, lanes 8e11).
very complex so as their role is not well characterized
[13,16,17] in spite of long-term study [47,48] and, in addition,
contradictory reports with cultures grown under anaerobic
and aerobic conditions on different carbon sources have been
published [48,49]; further study is required.
Note that the data obtained at pH 7.5 confirm the results
reported [9].
To understand the mechanisms of E. coli Hyd-enzymes
activity and regulation, H2 production was investigated during
glucose fermentation at a low pH. In contrast to pH 7.5 when
VH2 by the hyaB and hybC mutants as well as by the double hyaB
hybC mutant was similar to the wild type, at pH 6.5 and 5.5 VH2
by these mutants (but not by hybC at pH 5.5) was increased
significantly ( p < 0.001, Fig. 3). The increase might occur if Hyd-
1 and Hyd-2 are operating as H2 uptaking and oxidizing Hyd-
enzymes. The last statement is in favor with view point about
Hyd-1 and Hyd-2 activity mode under fermentation [8,13].
Moreover, the data of VH2 by hybC mutant at pH 5.5 could point
out a different role of Hyd-2 according to pH when Hyd-1 was
major in H2 uptake. Again, several explanations about different
role of hyaB and hybC mutants similar to those for glycerol
fermentation conditions (see above) might be given for these
findings. The results are in conformity with the results of
a similar pattern of increasing H2 production with decreasing
pH reported [26]. Hyd-3 more than Hyd-4 becomes responsible
for H2 production at a low pH; further study is required.
Importantly, Hyd-enzymes activity and expression at
different pH might depend on fermentation substrate (glycerol,
glucose) and other factors. A different level of H2 production
and a distinguishing expression and activity of Hyd-enzymes
have been reported in our lab [18,23] and by Rossman et al. [50]
and confirmed in Slonczewski lab [26]. Glucose (0.2%) or
formate (100 mM) used in the growth media [16,18,23,41] and Fig. 5 e Verification of E. coli JWD2689 (hycG) and SW1002
their absence [26] can attribute to this discrepancy; interaction ( fhlA hycG) (A) and of JWD2472 (hyfG) and SW1001 ( fhlA
between appropriate genes or between Hyd-enzymes may hyfG) (B) strains. For strains, see Table 1; for the others, see
contribute too. text. Four independent colonies of SW1001 were tested.
Please cite this article in press as: Trchounian K, et al., Escherichia coli hydrogenase activity and H2 production under glycerol
fermentation at a low pH, International Journal of Hydrogen Energy (2011), doi:10.1016/j.ijhydene.2010.12.128
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 1 ) 1 e9 7
3.5. Role of fhlA mutation and Hyd-3 and Hyd-4 in Eh Besides, in contrast to glycerol fermentation, during glucose
change and H2 production fermentation by fhlA strain and by fhlA hycG and fhlA hyfG
double mutants, H2 production was lowered at pH 6.5 and it was
A decrease in Eh down to strong negative values was observed less at pH 5.5 (not shown). At pH 6.5, VH2 by the double fhlA hycG
with E. coli JW2701 as well as fhlA hycG and fhlA hyfG double and fhlA hyfG mutants was w1.5-fold lower than that by the fhlA
mutants at pH 6.5 and 5.5 (see Fig. 1). This indicates a high strain (not shown). The results show a responsibility of Hyd-3
total Hyd-activity and H2 production under the conditions and Hyd-4 and suggest a role of the fhlA gene or appropriate
above. However, the kinetics of Eh decrease was different: it gene product, FhlA, in Hyd-3 and Hyd-4 expression and activity
was less with fhlA strain at pH 5.5 (see Fig. 1). in H2 production under glucose fermentation at a low pH. This is
VH2 was mentioned above to depend on pH. This was in conformity with data about Hyd-3 responsibility in H2
recently determined for fermentation conditions by Noguchi production during glucose fermentation at different pH; the
et al. [26]: unfortunately, fermentation substrate and other hycE gene (coding a large subunit of Hyd-3 [10]) expression was
conditions were not clarified but they may affect the demonstrated to be increased when pH decreased, whereas the
processes. Upon glycerol fermentation at pH 6.5, VH2 by fhlA activity of Hyd-3 was unchanged with a shift in pH [26].
strain as well as double fhlA hycG mutants did not demon-
strate large changes (Fig. 6), but at pH 5.5 it was lowered w2.3-
fold with the fhlA mutant and increased w2-fold with the 4. Concluding remarks
double fhlA hyfG mutant (comp. with wild type, see Fig. 2). The
results point out that the appropriate gene products, FhlA In this study, E. coli hydrogenase activity and H2 production
protein and Hyd-4 both, might affect H2 production and Hyd-4 were studied at a low pH. VH2 during glycerol fermentation at
can work in a reverse mode probably as H2 uptaking and pH 5.5 was shown to be w1.5-fold higher than that at pH 6.5 or
oxidizing Hyd-enzyme. Maeda et al. [28] have shown that E. above but less than that under glucose fermentation at
coli Hyd-3 operates as a reversible enzyme during glucose appropriate pH. It was inhibited by DCCD; however DCCD
fermentation at pH 6.8. Moreover, Trchounian and Trchou- inhibition of H2 production was increased with pH decreasing.
nian [9] have established reversible mode with Hyd-2 under DCCD inhibition of Hyd-enzymes is interesting to understand
glycerol fermentation but at pH 7.5 (see Fig. 4). As noted, two- a role of Hþ transport through the bacterial membrane
directional Hyd-2 has been also suggested for different including that via F0F1, since defects in F0F1 might stimulate H2
conditions by Lukey et al. [17]. In addition, these results production at pH 7.5 [9].
suggest that each Hyd-enzyme operating in one direction H2 production study with hyaB and hybC mutants with
might change its direction depending on pH and carbon defective Hyd-1 and Hyd-2, correspondingly (see Fig. 3),
source. Hyd-4 functioning upon glycerol fermentation at a low suggests that during glycerol fermentation at pH 6.5, Hyd-1
pH could be unexpected novel finding in addition to that with and Hyd-2 were working in a reversed, non-H2 producing
the operation of this enzyme upon glucose fermentation at pH mode. Under glycerol fermentation at pH 6.5, H2 production by
7.5 [18,23] (see Fig. 4). fhlA strain as well as double fhlA hycG and fhlA hyfG mutants at
Interestingly, DCCD effectively inhibited H2 production by a low pH (see Fig. 6) indicate that the appropriate gene prod-
fhlA strain as well as double fhlA hycG and fhlA hyfG mutants in ucts FhlA and Hyd-4 both affect H2 production and Hyd-4 can
spite of different values for VH2 (Fig. 6). This did not add operate in a reverse mode.
anything novel to the DCCD inhibition findings discussed In contrast to glycerol fermentation, upon glucose
above for wild type. fermentation H2 production by fhlA strain as well as double
fhlA hycG and fhlA hyfG mutants was lowered at pH 6.5; it was
less at pH 5.5 (see Fig. 6). The results show a responsibility of
Hyd-3 and Hyd-4 and suggest a role of fhlA gene or FhlA in
regulation of Hyd-3 and Hyd-4 activity for H2 production under
glucose fermentation at a low pH; this might be due to
appropriate transcription activation.
Followed from this and different groups [9,17,28] study it
could be concluded that reversibility is likely to be a property
of Hyd-enzymes having a key role in regulation of hydrogen
metabolism under different environment. This would be
important for construction of new mutants, stimulating H2
production and biotechnological application of glycerol.
Acknowledgments
Fig. 6 e VH2 and effect of DCCD for E. coli mutants with Thanks to Dr. A. Poladyan and A. Pogosyan for help in some
absent FhlA, defective Hyd-3 and Hyd-4 during glycerol measurements. The study was supported by the Grant #1012-
fermentation at different pH. For the others, see legends to 2008 from the Ministry of Education and Science of the
Figs. 1 and 2. Republic of Armenia.
Please cite this article in press as: Trchounian K, et al., Escherichia coli hydrogenase activity and H2 production under glycerol
fermentation at a low pH, International Journal of Hydrogen Energy (2011), doi:10.1016/j.ijhydene.2010.12.128
8 i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 1 ) 1 e9
Please cite this article in press as: Trchounian K, et al., Escherichia coli hydrogenase activity and H2 production under glycerol
fermentation at a low pH, International Journal of Hydrogen Energy (2011), doi:10.1016/j.ijhydene.2010.12.128
i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y x x x ( 2 0 1 1 ) 1 e9 9
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Please cite this article in press as: Trchounian K, et al., Escherichia coli hydrogenase activity and H2 production under glycerol
fermentation at a low pH, International Journal of Hydrogen Energy (2011), doi:10.1016/j.ijhydene.2010.12.128